Computational simulations of identified marine-derived natural bioactive compounds as potential inhibitors of oral cancer

Oral squamous cell carcinoma is characterized by the upregulation of RAC-alpha serine/threonine-protein kinase (Akt1) and RAC-beta serine/threonine-protein kinase (Akt2). In this work, Akt1 and Akt2 were inhibited using a cocktail of 20 marine algae chemicals. From the PyRx Virtual Screening Tool, dieckol, 6,6′-bieckol, siphonaxanthin and sargachromanol E were chosen as the best four compounds for Akt1 based on the scoring. Similarly, dieckol, 6,6′-bieckol, dioxinodehydroeckol and caulerpenyne were chosen as Akt2 inhibitors. Additionally, the results of the Lipinski rule of five indicated that some of the selected compounds, such as dieckol, 6,6′-bieckol and siphonaxanthin, violated some Lipinski rules, but they demonstrated excellent binding in terms of scoring. Thus, this study demonstrates that the identified lead compounds may act against Akt1 and Akt2 in oral cancer.

(Bcl-xL) protein, hence inhibiting the apoptotic process. AKT also inhibits caspases directly implicated in apoptosis and FOXO-1, a transcription factor that regulates the production of proapoptotic genes such as Bcl-2-like protein 11 or Bim and Fas-ligand (FasL) [16,17]. AKT has also been shown to inhibit glycogen synthase kinase 3 (GSK-3) and FOXO activity, which results in the overexpression of cyclin D1 [18,19]. When cyclin D1 is activated, it results in an increase in the expression of cyclin-dependent kinase (CDK) 4 and 6, which allows cells to exit the G1 phase and enter the replication phase, that is, the S-phase, thereby increasing proliferation. Additionally, AKT modulates the cytoplasmic location of p27, which is critical for tumor aggressiveness and metastasis [20].    As the stimulation of Akt has been an important predictive factor in OSCC, blocking it may provide a viable molecular strategy for therapies [21]. Akt or protein kinase B (PKB) is composed of three closely related isoforms: Akt1, Akt2 and Akt3 (or PKBa/b/c, separately). Three Akt isoforms are structurally and functionally identical and have comparable activation methods, but their tissue expression sites are unique [22]. Akt1 and Akt2 are highly expressed in tissue, whereas Akt3 expression is restricted. The study focuses on Akt1 and Akt2 as potential targets for OSCC.
The marine environment has indeed been demonstrated to be a valuable source of compounds with unusual and distinct chemical properties that can be used to generate novel treatments with increased potency and specificity.
Oceans constitute around 70% of the earth's surface and provide habitats for a wide variety of creatures [23]. Numerous metabolic products are produced by these species. Lower organisms, in particular, produce a plethora of secondary metabolites that serve as "defense and attack" signaling molecules. These molecules, which come from a variety of chemical classes, have the potential to be used as healthcare therapies [24]. Bacteria, fungi, corals, microand macroalgae, gorgonians, sponges, nudibranchs, bryozoans, sea cucumbers, tunicates and sea hares, among other marine organisms, have yielded several promising therapies in recent decades [25]. Efforts have been made to isolate these chemicals, with more than 10,000 natural products (NPs) of potential biotechnological interest identified to date. Algae are one of the ocean's most valuable commodities, both commercially and ecologically [26]. The

Compound preparation
This study focused on 20 chemicals isolated from marine algae Table 1. The structures of the molecules were retrieved from the PubChem database in sdf format (https://pubchem.ncbi.nlm.nih.gov/). The chemicals were loaded into OpenBabel and subjected to energy minimization using the Python Prescription Virtual Screening Tool (PyRx)16. The conjugate gradient approach was used to minimize the energy in the Universal Force Field (UFF). The total number of steps was set to 200, while the number of updates was set to 1. Additionally, the reduction procedure was configured to terminate at an energy difference of less than 0.1 kcal/mol [27].

Protein preparation
The crystal structures of Akt1 (PDB ID: 3QKL) and Akt2 (PDB ID: -2JDO) were obtained from the Protein Data Bank ( https://www.rcsb.org/). Separation of the ligand from the protein was accomplished. The protein was supplied with polar hydrogen atoms and Kollman charges, while the water molecules were removed. The final generated file was reduced and saved in PDBQT format for further examination using the UCSF Chimera program [28].

Structure-based virtual screening
To identify a new, powerful lead drug for Akt1,2, structure-based virtual screening (SBVS) was performed on produced compounds utilizing docking simulations. The binding energies were calculated using PyRx AutoDock VINA. To begin, a grid box covering the active region of the crystal structure was created with an exhaustiveness  of 8 and set to output just the lowest energy pose. Finally, the data were analyzed using the Discovery Studio [29] and PyMOL [30] tools.

ADME prediction
The Lipinski filter (http://www.scfbio-iitd.res.in/sof tware/drugdesign/lipinski.jsp) has been used to determine the absorption, distribution, metabolism and excretion (ADME) of chosen natural compounds. An orally active medication must satisfy at least four of the five criteria for drug similarity, namely. molecular mass, cLogP, hydrogen donor and acceptor and molar refractive index [31].

Results & discussion
The sixth most common cancer type in the world is head and neck cancer, which includes OSCC. In south-central Asia and Central and Eastern Europe, OSCC is among the leading causes of illness and mortality. OSCC usually affects the tongue, oropharynx, lip, floor of mouth, gingiva, hard palate and buccal mucosa, in decreasing order [32]. Members of the aldo-keto reductase (AKR) superfamily reduce the toxic and carcinogenic impacts of multiple genotoxic or non-genotoxic organic compounds and protect mammalian cells, but AKR1C2 and AKR1C3 from this family induce cancer progression by meditating phosphogluconate dehydrogenase (PGD) conversion through the phosphatidylinositol 3-kinase (PI3K)/Akt signal pathways [33]. PI3K is required for Akt isoform activation. Signaling molecules in the phosphoinositide 3-kinase (PI3K)/Akt pathway have drawn the attention of numerous researchers as therapeutic targets for cancer treatment due to their critical role in the carcinogenesis of several malignancies, including OSCC [34]. All hallmarks of cancer and malignant aggressiveness, including anti-apoptosis, cell proliferation, angiogenesis, immortalization, tissue invasion and metastasis, are associated with PI3K/Akt pathway dysregulation [35].
Following a thorough examination of Akt1, Akt2 and OSCC, the goal of this research was to identify several potential lead compounds for OSCC. Twenty ligands from marine sources were evaluated for this study. This screening was conducted using molecular docking and other in silico techniques. In structure-based small molecule docking, a small drug molecule is inserted into the target protein's binding cavity and the resulting docking position is evaluated using a specific scoring scheme (binding affinity) [36]. Binding affinity is usually a top priority when choosing the best candidate for virtual screening. The lowest energy value reflects the strongest ligand protein binding, as evidenced by the best docking scores [37].
It is currently the greatest way to forecast docking conformations of compounds that are energetically favorable to bind with a pharmacological receptor sites quickly, and it has grown in prominence as a way to save time and money in the drug discovery and development pipeline. Thousands of compounds can be evaluated for possible pharmacological activity using docking studies at minimal cost and in a short period of time. In this study, the authors used the docking technique to uncover novel Akt1 and Akt2 inhibitors utilizing SBVS on marine-based compounds.
Protein, fiber, vitamins, polyunsaturated fatty acids, macroelements and trace elements are all found in abundance in algae. Lately, they have been discovered to be a good source of antioxidants, phycocolloids, proteins, vitamins, minerals, carotenoids, soluble dietary fiber, polyunsaturated fatty acids, phycobilins, polysaccharides, sterols, tocopherols, terpenes and phycocyanins, among other bioactive components. Apart from their potential application as therapeutic agents in the biomedical field, these compounds have been shown to have nutritional and functional benefits [38]. Algae's multifunctional properties should be fully explored due to their unique structures and biological characteristics. Furthermore, the fact that more than 30,000 algal species have been identified around the world supports the assumption that algae are a very prolific source of structurally distinct NPs with medicinal potential. Furthermore, after sponges, bacteria and phytoplankton, algae have been identified as a key source of novel marine chemicals. Twenty compounds from marine algae were chosen for virtual screening against the OSCC target proteins Akt1 and Akt2 in this investigation. The compounds were graded based on their binding energy after the docking investigations. The best four compounds for Akt1 were picked from this output based on the score parameters: dieckol, 6,6 -bieckol, siphonaxanthin and sargachromanol E. Akt2 inhibitors were chosen from dieckol, 6,6 -bieckol, dioxinodehydroeckol and caulerpenyne.
The binding affinities of selected compounds with Akt1 (PDB ID-3QKL) were found to be in the range of -9.5 kcal/mol to -10.8 kcal/mol, indicating a significant binding affinity. The binding affinities of Akt2 (PDB ID-2JDO) and marine-based chemicals were again found to be in the range of -9.2 kcal/mol to -11.4 kcal/mol, indicating a significant binding affinity. The Visualizer in Discovery Studio was used to visualize the the proteinligand interactions of all drugs with Akt1 and Akt2, respectively. Bonds formed among these amino acids of Akt1-ligand complexes as well as Akt2-ligand complexes are shown in Figures 1 & 2. 3D interaction of selected compounds with Akt1 and Akt2 is shown in Figures 3 & 4.
The dieckol-Akt1 complex had the strongest bonding of the four complexes, with a binding energy of -10.8 kcal/mol. It forms electrostatic and hydrogen bonds, which indicate a positive ligand-protein interaction, since these bonds are essential for forming a strong binding. In the realm of molecular chemistry, Pi-cation interactions play a crucial role [39][40][41]. In molecular recognition as well as chemical and biological catalysis, the pication bond is crucial. Akt1-dieckol Table 2 lists the amino acids implicated between the Akt-1-Dieckol interactions in detail. Dieckol, like Akt1, demonstrated a stronger affinity for Akt2 than the other chemicals, with a binding energy of -11.4. The docked complexes were seen in PyMOL, and all of the identified compounds attached to the same binding site of Akt1 and Akt2.
The projected ADMET characteristics were calculated to aid future lead optimization. Except for a few compounds, the Lipinski rule is followed by the majority of the compounds. Table 3 shows the ADME properties that were predicted.

Conclusion
The major goal of this pilot study was to see if SBVS could be used to find novel inhibitors with action against Akt1 and Akt2 in oral cancer. The SBVS was carried out with the help of AutoDock VINA, which is included in the PyRx 0.8 package. The findings also point to new study directions that could be explored in future initiatives. The study was done in silico with a variety of effective ways. The outcomes of these in silico approaches generate a precise and clear sense of the efficiency of known anti-tumor medicine by interacting with the Akt pathway and providing therapeutic efficacy.By interacting with the Akt pathway and giving therapeutic efficacy, the results of these in silico methodologies generate a precise and clear notion of the efficiency of the established antitumor medicine. The findings of this study pointed to a promising future for this medicine as a potent alternative to the already-proven Akt inhibitors in the treatment of OSCC. As a result, dieckol, 6,6 -bieckol, dioxinodehydroeckol, caulerpenyne, siphonaxanthin and sargachromanol E in the Akt pathway can be considered for therapy of OSCC.

Summary points
• Oral cancer is among the most difficult diseases that humanity faces, and despite numerous advances in the field of oral cancer detection and treatment, it continues to be a major public health problem worldwide. • The upregulation of RAC-alpha serine/threonine-protein kinase (Akt1) and RAC-beta serine/threonine-protein kinase (Akt2) is the main cause of oral squamous cell carcinoma. • The marine environment represents a unique source of bioactive compounds with high pharmaceutical potential.
• Algae are one of the ocean's most valuable commodities, both commercially and ecologically.
• Twenty compounds from marine algae were selected for screening against Akt1 and Akt2.
• The best 4 compounds for Akt1 were picked from this screening based on the score parameters: dieckol, 6,6 -bieckol, siphonaxanthin and sargachromanol E. Akt2 inhibitors were dieckol, 6,6 -bieckol, dioxinodehydroeckol and caulerpenyne. • The predicted absorption, distribution, metabolism and excretion showed that except for a few compounds, the Lipinski rule is followed by the majority of the compounds. • Hence, these compounds may act as potential leads for identifying a new oral cancer drug.

Author contributions
Equal contribution among all; added contribution in conceptualization by PM Natarajan.

Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.
No writing assistance was utilized in the production of this manuscript.

Open access
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